1. Field of the Invention
This invention relates to a fluorescence observation or fluorescence measuring system, and a fluorescence observation or fluorescence measuring method.
2. Description of Related Art
By the developments in recent years of measuring instruments and apparatuses in the fields of microscopes, fluorescence microscopes, and protein and DNA analytical apparatuses, tendencies of observations and measurements in these fields are changed. In the changes of the tendencies, there are two great currents described below.
One of them is a change of observation-measurement objects from the observations and measurements of fixed cells to those of living cells. The advent of the post-genome era has increased the importance of the technique that allows accurate observation and measurement of feeble fluorescent light in a broad band with respect to fluorochrome single-molecule fluorescence measurement and a simultaneous analysis of functions of living bodies by the color diversification of fluorochrome. In particular, in the most advanced research field, the need that observations of cells, in vivo, should be continued for a long period of time (in the range from several days to a few weeks) has recently been increased for purposes of the functional clarification of living bodies and the behavior analysis and interaction clarification of proteins, and various techniques of such observations have been developed. In order to observe the cell, the technique of producing a fluorescent protein in a desired cell or introducing the fluorochrome to observe fluorescent light emanating therefrom is often used. The latest technique involves a single-molecule fluorescence observation that is thought of as an ultimate feeble-fluorescence observation and trends to feebler-fluorescence observation and measurement. In a general fluorescence observation, when light for exciting a fluorescent substance (excitation light) is too intense, damage is caused to the cell. Thus, in order to keep the cell alive for a long period of time, it is necessary to set as low an intensity of the excitation light as possible. It is known that when the fluorescent substance is irradiated with the excitation light, the fluorescent light is bleached, not to speak of the cell observation. Even for the purpose of suppressing bleaching by irradiating the substance with faint excitation light, it is very useful to enable an image with a good S/N ratio to be observed with feeble fluorescent light.
When faint excitation light is used, however, the intensity of fluorescent light to be detected is lowered and it becomes difficult to obtain an image with a high S/N ratio. As the fluorescent light becomes feeble, including the case of the single-molecule fluorescence observation that is the ultimate feeble-fluorescence observation, a contribution to the noise increases and the S/N ratio is reduced. Here, the noise refers to auto-fluorescence mainly emanating from an optical system or a specimen.
The other is a change from an apparatus provided with only the function of observation like a conventional microscope apparatus to an apparatus further provided with a means for measuring and quantifying the intensity of fluorescent light, the wavelength, and the localization of matter to be detected. Accurate quantification, including the noise, has been required.
In a fluorescence observation apparatus such as a fluorescence microscope, and a fluorescence measuring apparatus such as a genome/protein analytical apparatus, various wavelengths are observed and measured in a wide range from ultraviolet to infrared. In particular, the fluorescence observation and measurement by three excitations, called U excitation, B excitation, and G excitation, respectively, are typical. The U excitation is caused at a wavelength of about 365 nm and fluorescent light of wavelength about 450 nm is observed and measured; the B excitation is caused at a wavelength of about 488 nm and fluorescent light of wavelength about 540 nm is observed and measured; and the G excitation is caused at a wavelength of about 550 nm and fluorescent light of wave-length about 600 nm is observed and measured.
Conventional fluorescence observation apparatuses and fluorescence measuring apparatuses are proposed, for example, in Japanese Patent Kokai Nos. Hei 08-320437 and Hei 08-178849.
A conventional microscope for fluorescence observation is set forth, for example, in Japanese Patent Kokai No. 2001-83318, as the microscope constructed so that the fluorescence observation by ordinary reflecting illumination and that by totally reflecting illumination are switched to each other.
A fluorescence detecting system through the conventional fluorescence microscope such as the microscope set forth in Kokai No. 2001-83318 is constructed so that a fluorescent substance is irradiated with excitation light to detect fluorescent light emanating from the fluorescent substance through a detector and thereby a specimen is observed.